Pseudomonas aeruginosa keratitis is a sight-threatening complication of contact lens wear. Ordinarily, the ocular surface is effective against microbial infiltration through a variety of mechanical, anatomical, and immunological defense mechanisms which protect the cornea . However, contact lens wear, ocular injury and/or surgery predispose individuals to bacterial keratitis. In the case of contact lens wear, the Gram negative bacterium P. aeruginosa is the most frequently isolated causative agent. As the number of antimicrobial compounds effective against P. aeruginosa decreases, due to of the acquisition and spread of antibiotic resistance, there is a growing need for novel therapeutic approaches corneal infection. The fact that P. aeruginosa can cross the corneal epithelium into the stroma to cause disease in contact lens wearers suggest a compromise in host defense. However, little is known of the mechanisms by which bacteria reach the stroma after adherence to corneal epithelial cells and how contact lenses increase the incidence of P. aeruginosa infection. Preliminary data show that bacteria initially attempting to penetrate corneal epithelium die in the process, suggesting killing by epithelial antimicrobial factors. However, they also show that at later time points, viable bacteria do penetrate the epithelium, and are then better able to penetrate through multiple layers of epithelial cells. This suggests that bacterial adaptation is required for virulence. In vivo data using bacterially contaminated contact lenses show that disease onset follows a delayed course, (beginning only after 1-2 weeks), with P. aeruginosa biofilms consistently found on lenses involved in infections. This proposal focuses on factors that modulate translocation of P. aeruginosa through corneal epithelia. The hypothesis to be tested is that under normal circumstances antimicrobial peptides expressed by the corneal epithelium limit P. aeruginosa translocation of that epithelium, but that prolonged exposure to corneal epithelia induces differential bacterial gene expression which enhances translocation capacity. Preliminary data show that bacteria acquire increased capacity to translocate. Microarray analysis will be used to compare gene expression in translocated bacteria versus those naive to translocation to determine what changes have occured. Established in vitro and in vivo models will then be used to determine the role(s) of differentially expressed genes in modulating P. aeruginosa translocation of corneal epithelia.